The present invention relates to a microfluidic device for in vitro 3D cell culture experimentation comprising a body in which is provided a cell culture chamber at least partly filled with a scaffolding substance for maintaining a cell culture, and a fluid path communicating with the cell culture chamber for directing a fluid stream along the scaffolding substance. The present invention further relates to a method for in vitro 3D cell culture experimentation, including complex living tissue reconstruction, using the microfluidic device of the present invention.
In vitro cell culture experimentation is important in biological and medical sciences for allowing investigation of cellular behavior of individual cells or of cells as part of larger cell cultures. For instance the investigation of uptake of biomolecules by cells may lead to improved knowledge and understanding of the effect of such biomolecules on a cellular, tissue, organ and subject level, which in turn may lead for example to the development of personalized medicine. Currently pharmacokinetic and toxicological evaluation of drug candidates relies largely on costly, labor-intensive, time-consuming and ethically questionable animal test systems, which show only very limited predictive value for clinical efficacy and toxicity.
Many methods and devices for culturing, expanding and differentiating cells in vitro have thus been developed. A conventional and still often used method is growth and maintenance of cells or cell cultures on a suitable growth surface such as a cell culture dish filled with liquid or jellified culture medium. The culture medium may comprise specific constituents which affect the growth and maintenance of the cells or cell culture in desired ways. However the predictive value of these two dimensional (2D) cell culture models for some application may be still very limited, because of the loss of physiological context.
With 3D scaffolds, for example cells incapsulated in a scaffolding substance such as hydrogel, tissue-like connectivity may be achieved, but there are limits in controlling the cell culture conditions. The 3D models mostly lack the complexity required for pharmacokinetic studies. For many applications in such models there is a limited nutrient supply to the cell culture and an accumulation of metabolic waste products that can confound cell responses to drugs. The 3D models also fail to mimic spatiotemporal biochemical gradients existing in vivo, and lack the provision of mechanical cues such as flow, perfusion, pressure, mechanical stress. It is also problematic for real-time imaging, and biochemical analysis can hardly be performed in live cells due to reaction-diffusion phenomena. Furthermore, it is not easily possible to engineer microsystems that integrate multiple organ/tissue mimetics with active vascular conduits and barrier tissues.
Microfluidic devices such as microfluidic chips allow for addressing these limitations. With microfluidic devices fluid flow may be controlled in the micrometer and nanoliter scale in precisely defined geometries. Because of the micro geometrical dimensions, the flow of fluids is laminar, and placement of fluid volumes in very low amounts is possible. The ability of exactly timing fluid flow allows precise chemical and physical control of the microenvironment. For cell cultures in microfluidic devices the doses delivered to cells can be measured in nanoliters or less, representing a significant improvement in precision. Small volume effects of fluids mimic physiological conditions of cells or cell-populations in tissues more appropriately than cells that are cultured in larger volumes. Microfluidic systems also allow detailed analysis of cell migration in a social context. Controlling the spatiotemporal cues of the microenvironment and the ability to shape the geometry of cultured cells for instance allows studying of primary neuronal cells and cell lines in microfluidic chips.
Integration of microfluidics with 3D scaffolding systems renders it possible to adapt culture conditions both biochemically and biomechanically, such as creating dynamic 3D structures, and provides a microenvironment that allows formation of artificial tissues from cultured cells. Microfluidic cell culture devices allow precise control of cell numbers and cell density in a given area or volume, and can provide placement of cells in complex geometries. Because cells can be organized into three-dimensional geometries in scaffolding substances such as hydrogels in the microfluidic devices, it is possible to culture cells in 3D structures resembling those in tissues. Homotypic tissue culture models may be achieved in microfluidic devices as well as heterotypic tissue culture models that mimic the respective tissue closely both from a histologic as well as from a physiological and functional standpoint. This allows for instance for high-throughput pharmacological studies and might result in using microfluidic cell culture systems also for regenerative purposes.
The small dimensions of spatially separated microfluidic compartments in microfluidic cell culture devices allow assembly of a multitude of individually controllable cell cultures in chambers on a single device. This facilitates high parallelization of experiments, high throughput of samples and reactions and thus improvement of reproducibility, as well as a reduction in reagent costs.
Resulting from the above-mentioned advantages, microfluidics has become particularly valuable for analysis of single cell dynamics. With the help of microfluidic devices cell growth and regulation of cell size can be directly observed and lineages of single cells can be tracked for several generations. On a molecular level microfluidics allow the characterization of transcription factor and gene expression dynamics in single-cells thereby adding substantially to our understanding of the function of biological systems.
The presently available microfluidic devices for in vitro 3D cell culture experimentation comprise a closed system to shield the cell culture and the culture conditions from possible outside influences, and provide a limited accessibility to the cell culture grown in the culture chamber of the device. Thus the known devices render the simultaneous manipulation and analysis of cultured cells rather difficult, particularly monitoring of cells in complex geometries with high spatial and temporal resolution and their individual retrieval during or following experiments.
As a result, there is a need for an improved microfluidic system for cell culture investigation which may be particularly applied in drug studies, vaccine development and other types of medical research. The present invention thus provides a new device and method for cell culture investigation, with which it is possible to investigate all types of cells such as vascular cells and organ cells individually or in functional units of tissues and organs in vitro. These and other aspects of the invention are evident from the specification and claims hereinafter.